Polynucleotide capture materials, and methods of using same

ABSTRACT

Methods for processing polynucleotide-containing biological samples, and materials for capturing polynucleotide molecules such as RNA and/or DNA from such samples. The RNA and/or DNA is captured by polyamindoamine (PAMAM (Generation 0)) bound to a surface, such as the surface of magnetic particles. The methods and materials have high efficiency of binding RNA and of DNA, and of release, and thereby permit quantitative determinations.

CLAIM OF PRIORITY

This application is a continuation application of application Ser. No.13/692,980, filed on Dec. 3, 2012, which is a continuation ofapplication Ser. No. 12/172,214, filed on Jul. 11, 2008 (now U.S. Pat.No. 8,324,372), which claims the benefit of priority under 35 U.S.C.§119(e) to U.S. provisional application Ser. No. 60/959,437, filed Jul.13, 2007, all of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Technical Field

The technology described herein generally relates to methods forprocessing biological samples, and more particularly relates tomaterials for capturing polynucleotide molecules such as RNA and DNAfrom such samples, and permitting quantitative determination thereof.

2. Field of the Invention

The analysis of a biological sample such as a clinical sample or a testsample of food, for presence of a pathogen such as a virus, or todetermine the presence of a particular gene, will typically includedetecting one or more polynucleotides present in the sample. One type ofdetection is qualitative detection, which relates to a determination ofthe presence or absence of a target polynucleotide and/or thedetermination of information related to, for example, the type, size,presence or absence of mutations, and/or the sequence of the targetpolynucleotide. Another type of detection is quantitative detection,which relates to a determination of the amount of a particularpolynucleotide present in the sample, expressed for example as aconcentration or as an absolute amount by weight or volume. Detectionmay also include both qualitative and quantitative aspects. Quantitativedetection is typically, however, a more challenging pursuit than is asimple qualitative determination of presence or absence of apolynucleotide.

Detecting polynucleotides often involves the use of an enzyme. Forexample, some detection methods include polynucleotide amplification bypolymerase chain reaction (PCR) or a related amplification technique.Other detection methods that do not amplify the polynucleotide to bedetected also make use of enzymes. However, the functioning of enzymesused in such techniques may be inhibited by the presence of materials(known as inhibitors) that accompany the polynucleotide in manybiological—particularly clinical—samples. The inhibitors may interferewith, for example, the efficiency and/or the specificity of the enzymes.

Polynucleotide detection today is moving towards ever more rapid, andever more sensitive techniques. For example, rapid and accuratediagnosis of viral infections is invaluable for accurate patientmanagement by directing the administration of appropriate antiviraltherapy, eliminating the unnecessary utilization of antibiotics andmonitoring individual response to the prescribed regimen. Given itssignificant advantages of sensitivity, specificity and time to result,polynucleotide detection (or nucleic acid testing) has become thepresumptive international standard for viral diagnosis.

However, the application of nucleic acid testing to routine diagnosis ofviral targets has been limited to large clinical reference labs andmajor hospital labs due to the high cost, complexity and skill levelrequirements for implementing such testing. While significantimprovements have been made in recent years, the successful detection ofRNA viruses in particular requires extremely laborious extractionprocedures frequently relying on the use of toxic chemicals.Furthermore, RNA molecules can be very unstable and hence can requiredelicate processing/handling during their determination. These issues todate have been overcome with the use of large, expensive, time consumingrobotic equipment.

With the current demands on practice of medicine, laboratories thatcarry out diagnostic testing on patient samples see substantial benefitsfrom having extremely high throughput, which in itself is assisted ifthe time to arrive at a diagnostic outcome for a given sample is made asshort as possible. Testing may also be made more rapid if the actualsample on which the tests are run is made as small as possible. Morerecently, there has been a growing need for a small, easy to use,low-cost, automated platform for the extraction of high quality RNA fromviral targets in clinical specimens.

Correspondingly, then, the need to be able to isolate minute quantitiesof polynucleotides from complex biological samples in a manner thateffectively avoids the presence of, or reduces the detrimental impactof, inhibitors is ever more important. Furthermore, given theavailability of various stand-alone automated amplification apparatuses,it is desirable to be able to routinely and reliably extract from a rawclinical sample a quantity of polynucleotide that is ready—in terms ofpurity and quantity—for amplification.

The discussion of the background herein is included to explain thecontext of the technology. This is not to be taken as an admission thatany of the material referred to was published, known, or part of thecommon general knowledge as at the priority date of any of the claimsfound appended hereto.

Throughout the description and claims of the specification the word“comprise” and variations thereof, such as “comprising” and “comprises”,is not intended to exclude other additives, components, integers orsteps.

SUMMARY OF THE INVENTION

The process and materials herein are applicable to a number of testingtargets, in particular those that are RNA based, such as Influenza (A &B), RSV, HSV, CMV, Adenovirus, and Enterovirus.

The technology herein provides excellent RNA—as well as DNA—capture andrecovery via use of micro-particles having a high RNA and DNA bindingcapacity, such as 100 μg/mg beads, and a >90% release efficiency. Inexemplary embodiments, 8-10 μg RNA can be extracted from an overnightculture. Processes, as described herein, permit very fast (15-20 minutesincluding lysis) RNA extraction from cellular or viral material, via asingle tube process. Processes, as described herein, comprise astreamlined procedure having fewer steps (such as six) to proceed fromraw sample to purified RNA. Such processes therefore provide anextremely effective clean-up of RNA from raw biological samples, therebypermitting PCR to be performed thereon. The methods and processes areapplicable across a wide variety of sample matrices, as well as clinicalbuffers used when collecting raw samples, e.g., M4, UTM, and Todd HewitBroth.

Suitable targets, that have assays used in clinical testing, and thatmay be the subject of sample preparation processes as described herein,include, but are not limited to: Chlamydia Trachomatis (CT); NeisseriaGonorrhea (GC); Group B Streptococcus; HSV; HSV Typing; CMV; Influenza A& B; MRSA; RSV; TB; Trichomonas; Adenovirus; Bordatella; BK; JC; HHV6;EBV; Enterovirus; and M. pneumoniae.

One aspect of the present invention relates to a method for processingone or more RNA and/or DNA compounds (e.g., to concentrate the RNAand/or DNA compound(s) and/or to separate the RNA and/or DNA compound(s)from inhibitor compounds (e.g., hemoglobin, peptides, faecal compounds,humic acids, mucousol compounds, DNA binding proteins, or a saccharide)that might inhibit detection and/or amplification of the RNA and/or DNAcompounds).

In some embodiments, the method includes contacting the samplecontaining the RNA and/or DNA compounds and PAMAM (Generation 0) thatpreferentially associates with (e.g., retains) the RNA and/or DNAcompounds as opposed to inhibitors. The PAMAM (Generation 0) istypically bound to a surface (e.g., a surface of one or more particles).The PAMAM (Generation 0) retains the RNA and/or DNA compounds so thatthe RNA and/or DNA compounds and inhibitors may be separated, such as bywashing the surface with the compound and associated RNA and/or DNAcompounds. Upon separation, the association between the RNA and/or DNAcompound and the PAMAM(Generation 0) may be disrupted to release (e.g.,separate) the RNA and/or DNA compounds from the compound and surface.

The present disclosure provides for a method for isolatingpolynucleotides from a cell-containing sample, the method comprising:contacting the sample with a lysis solution and a plurality of bindingparticles coated in PAMAM(Generation 0), so that the polynucleotides areliberated from the cells and become bound to the PAMAM(Generation 0),thereby creating binding particles bound with polynucleotides and asolution containing residual cellular matter; compacting the bindingparticles bound with polynucleotides; removing the solution containingresidual cellular matter; washing the binding particles; and releasingthe polynucleotides from the binding particles.

The present disclosure further includes a process for concentrating RNAfrom a sample containing polymerase chain reaction inhibitors, themethod comprising: contacting between 500 μl and 1 ml of the sample witha plurality of RNA binding particles, the binding particles configuredto preferentially retain the RNA in the sample as compared to thepolymerase chain reaction inhibitors; concentrating the plurality ofparticles having the one or more polynucleotides bound thereto into aneffective volume between 50 nanoliters and 5 microliters; and releasingthe one or more polynucleotides into <30 μl of solution.

The present disclosure still further includes a composition comprising:carboxyl modified microparticles; and PAMAM(Generation 0) bound via oneor more amine groups per molecule to one or more of the carboxylic acidgroups on the microparticles.

The present disclosure additionally includes a kit, comprising: a numberof sealed tubes, each containing lysis buffer; a tube containinglyophilized microparticles having PAMAM(Generation 0) bound thereto; atube containing liquid wash reagents, sufficient to analyze the numberof samples; and a tube containing liquid release reagents, sufficient toanalyze the number of samples, wherein each component of the kit isstored in an air-tight container.

The present disclosure still further includes a kit, comprising: a firstair-tight pouch enclosing a number of tubes, each tube containinglyophilized microparticles having PAMAM(Generation 0) bound thereto; asecond air-tight pouch enclosing a number of reagent holders, eachholder comprising: a tube containing liquid lysis reagents; a tubecontaining liquid wash reagents; and a tube containing liquid releasereagents.

The present disclosure additionally includes a method of making apolynucleotide retention member, the method comprising: washing aquantity of microspheres with carbonate and MES buffer; preparingsulfo-NHS and EDAC; incubating the microspheres with sulfo-NHS and EDACfor 30 minutes; washing the microspheres with MES and borate buffer;contacting the microspheres with PAMAM(0) for 8-10 hours; and rinsingunbound PAMAM(0) from the microspheres.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematically a typical process as described herein.

FIG. 2 shows schematically the action of DNA affinity beads as furtherdescribed herein.

FIG. 3 shows PCR curves for EV13 RNA extraction from nasal swabs.

FIG. 4 shows PCR curves for EV13 RNA extraction in M4 media.

FIG. 5 shows a comparison of PCR curves from RNA extracted from a bufferto one obtained from plasma.

FIG. 6 shows RNA extraction using beads.

FIG. 7 shows RNA extraction from plasma.

FIG. 8 illustrates extraction sensitivity.

FIG. 9 shows a flow-chart for a process of making PAMAM(Generation 0)coated microparticles.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

Analysis of biological samples often includes determining whether one ormore polynucleotides (e.g., a DNA, RNA, mRNA, or rRNA) is present in thesample. The technology described herein has applicability to determiningboth RNA and DNA that is present in a sample. For example, a sample maybe analyzed to determine whether the RNA of a particular pathogen ispresent, and also whether DNA of another or the same pathogen ispresent. If present, the RNA or the DNA may together or separately beindicative of a corresponding disease or condition.

Accordingly, the technology described herein is directed to materialsthat bind polynucleotides, and use of such materials in isolatingpolynucleotides, such as DNA and RNA, from biological samples. Thematerials, in conjunction with methods of using the materials, providefor rapid and reliable extraction of RNA and DNA from many differenttypes of biological samples, including quantitative determination ofboth the RNA and the DNA. Such methods are typically referred to as“sample preparation” methods. What is meant by such a term is theliberation, extraction, concentration, and/or isolation, of RNA and/orDNA of a target organism from a raw sample—such as obtained directlyfrom a patient or an agricultural or food product—where the raw samplecontains the target RNA and/or target DNA bound in cellular form. Theliberated target RNA and/or target DNA is placed, at the culmination ofthe process, in a form suitable for amplification and/or detection.

The terms DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), andtogether as polynucleotides, as used herein can mean an individualmolecule or population of molecules, such as identifiable by having aspecific nucleotide sequence common to all, or can mean collectivelymolecules of DNA or RNA having different sequences from one another. Forexample, a biological sample from a human patient may contain DNA fromthe patient's cells, having one sequence, and DNA or RNA from cells of apathogen, having a different sequence from that of the patient's DNA.The sample is thus referred to as containing DNA and RNA (or, together,polynucleotides), even though there are molecules of DNA (or RNA) in thesample that are different (chemically distinct) from one another. Themethods herein can be used to liberate, collectively, molecules of DNAand RNA from both the patient's and the pathogen's cells in such asample. Typically, however, in such an instance, it will usually be theDNA or RNA of the pathogen that will be of interest, and which will beselectively amplified from amongst all the DNA and RNA that isultimately isolated from the sample. The DNA and RNA that is best suitedfor extraction by the methods herein has a size less than 7.5 Mbp,though it would be understood that larger DNA and RNA molecules may besusceptible to extraction and detection by the methods herein.

Typically, biological samples are complex mixtures. For example, asample may be provided as a blood sample, a tissue sample (e.g., a swabof, for example, nasal, buccal, anal, or vaginal tissue), a biopsyaspirate, a lysate, as fungi, or as bacteria. The RNA and/or DNA to bedetermined is normally contained within particles (e.g., cells such aswhite blood cells, or red blood cells), tissue fragments, bacteria(e.g., gram positive bacteria, or gram negative bacteria), fungi, orspores. One or more liquids (e.g., water, a buffer, blood, blood plasma,saliva, urine, cerebral spinal fluid (CSF), or organic solvent) istypically part of the sample and/or is added to the sample during aprocessing step. The materials and methods described herein arecompatible with a variety of clinical matrices, at least includingblood, urine, CSF, swab, and plasma.

Methods for analyzing biological samples include releasing RNA and/orDNA from the particles (e.g., bacteria) in the sample, amplifying one ormore of the released RNA and/or DNA (e.g., by polymerase chain reaction(PCR)), and determining the presence (or absence) of the amplifiedpolynucleotide(s) (e.g., by fluorescence detection).

Clinical samples present a variety of challenges especially in thedetection of target RNA and DNA through PCR or similar technologies. Atarget nucleic acid could be present in a concentration as low as 10copies per milliliter as measured against a background of millions orbillions of copies of competing nucleic acids (such as from a patient'snormal cells). Moreover, a variety of other biochemical entities presentin the clinical sample inhibit PCR. The inhibitors may also frustrateisolation of RNA or DNA from the sample, such as by being captured by amaterial designed to retain the RNA or DNA. If the concentration ofinhibitors is not reduced relative to the RNA or DNA to be determined,the analysis can produce false negative results. Examples of theseinhibitors, dependent upon the biological sample in question, arecellular debris such as membrane fragments, humic acids, mucousalcompounds, hemoglobin, other proteins such as DNA binding proteins,salts, DNAases, fecal matter, meconium, urea, amniotic fluid, blood,lipids, saccharides, and polysaccharides. For example, such inhibitorscan reduce the amplification efficiency of DNA and RNA by PCR and otherenzymatic techniques for determining the presence of DNA and RNA.

Therefore, an effective sample preparation method should lead to aconcentration of the target RNA or DNA, and should minimize presence ofinhibitory substances. The methods described herein may increase theconcentration of the DNA and/or RNA to be determined and/or reduce theconcentration of inhibitors relative to the concentration of DNA and/orRNA to be determined.

In addition, cells of some target organisms, such as gram positivebacteria (e.g. Group B Strep), are very hard to lyse, meaning thatlysing conditions can be very severe. Such organisms may requireadditional chemicals for lysing, such as mutanolysin, and may alsorequire higher temperatures for optimal lysis. Such conditions may beaccommodated by the materials and methods described herein.

Sample Preparation Process

A typical sample preparation process may be carried out in a processingchamber that includes a plurality of particles (e.g., beads,microspheres) configured to retain RNA and/or DNA of the sample under afirst set of conditions (e.g., a first temperature and/or first pH) andto release the RNA under a second set of conditions (e.g., a second,higher temperature and/or a second, more basic, pH), and to release DNAunder a third set of conditions (e.g., a third, different temperatureand/or a third, more basic, pH than that used in the first and secondconditions). Typically, the DNA and RNA are retained preferentially ascompared to inhibitors that may be present in the sample.

An exemplary sample preparation process is illustrated in FIG. 1. Thevarious reagents referred to in connection with FIG. 1 are described infurther detail elsewhere herein. At 100, a process tube 101, such as astandard laboratory 1.7 ml microcentrifuge tube, contains a biologicalsample comprising a liquid 109, such as an aqueous solution, andcellular materials 111, wherein at least some of the cellular materialsmay contain RNA and/or DNA of a target of interest. The biologicalsample may be any of those described elsewhere herein, and process tube101 may be any tube or suitable vessel, as further described herein. Itis to be understood that, although the process is illustrated withrespect to FIG. 1, the process is not limited to be carried out in atube. The sample and various reagents may be, for example, delivered to,and mixed and reacted within, chambers of a microfluidic device such asa microfluidic cartridge, as further described in U.S. patentapplication Ser. No. 11/281,247, filed Nov. 16, 2005 and incorporatedherein by reference.

A first pipette tip 103 contains a solution 107 of microparticles 105,that are delivered to the process tube and contacted with the biologicalsample contained therein. The surfaces of particles 105 are modified tohave PAMAM(0) attached, as further described herein, so that they retainRNA and/or DNA in preference to inhibitors in solution. Solution 107 maybe a lysis solution, as further described herein. The lysis solution maycontain a detergent, in addition to various enzymes, as describedelsewhere herein. Thorough mixing of the microparticles, the solution,and the biological sample may occur simply by turbulent combination ofthe two solutions upon release of the microparticle containing solutionfrom the pipette tip, or may occur via mechanical or manual agitation ofprocess tube 101.

First pipette tip 103 is positioned above process chamber 101, such asby manual operation by a user, or such as by an automated pipettinghead, an example of which is described in U.S. provisional patentapplication Ser. No. 60/959,437, filed Jul. 13, 2007, which isincorporated herein by reference.

At 110, using the same process tube 101, the microparticles, biologicalsample, and lysis reagents are incubated, such as by applying heat froman external source, as shown, so that the cells in the biological sampleare lysed, and liberate RNA and/or DNA. Under these conditions, the DNAmolecules bind to suitably configured surfaces of the micro-particles,as further described herein. Typically, the particles retain RNA and/orDNA from liquids having a pH about 9.5 or less (e.g., about 9.0 or less,about 8.75 or less, about 8.5 or less). It is to be noted that thebinding of DNA to the affinity microparticles happens concurrently withthe lysis process, and the binding is not adversely affected by thepresence of detergents and, in some instances, lytic enzymes in thelysis solution. The choice of temperature is dictated by what isrequired to lyse the cells in question, and heat is not required toeffectuate binding of the RNA or DNA to the particles. Typically, thosecells having tougher cell walls (e.g., lysteria, or anthrax) willrequire higher temperatures. For example, Chlamydia determinationutilizes a temperature of 37° C. for a duration of 5-10 minutes forlysis and binding, whereas Group B Streptococcus determination utilizesa temperature of 60° C. for a duration of 5-10 minutes. Generally, theliquid is heated to a temperature insufficient to boil liquid in thepresence of the particles.

At 120, the microparticles are concentrated or compacted, and theremaining solution containing residual cellular matter 125 is removed,for example by a second pipette tip 123. By compacted is meant that themicroparticles, instead of being effectively uniformly distributedthrough a solution, are brought together at a single location in theprocess tube, in contact with one another. Where the microparticles aremagnetic, compaction of the microparticles may be achieved by, forexample, bringing a magnet 121 into close proximity to the outside ofthe process chamber 101, and moving the magnet up and down outside thechamber. The magnetic particles are attracted to the magnet and aredrawn towards the inside of the wall of the process chamber adjacent themagnet.

Pipette tip 123 removes as much of the remaining solution (sometimesreferred to as supernatant, or solution having residual cellular matter)as is practical without drawing up significant quantities ofmicroparticles. Typically a pipette tip may slide into process chamber105 without contacting the microparticles. In this way themicroparticles are concentrated, by being present in a smaller volume ofsolution than hitherto. Pipette tip 123 may be a different tip frompipette tip 103, or may be the same tip. In some embodiments, afterremoval of the solution containing residual cellular matter, less than10 microliters of solution is left along with the particles. Typicallythis is achieved by both compaction of the microparticles to a smallpellet, but also positioning that pellet away from the location whereinthe pipette will be introduced for removal of the supernatant. Thepositioning of the pipette in relation to the bottom of the tube is alsoimportant so that almost all of the supernatant is removed. The pipettetip should be almost close to the bottom of the tube (within 1-2 mm) butwithout completely sealing the pipette tip against the tube bottom. Astellated pattern may also be used at the bottom of the lysis tube, (asdescribed in U.S. provisional patent application Ser. No. 60/959,437,filed Jul. 13, 2007, and incorporated herein by reference), but thepositioning of the patterns in relation to the location of the magnetbecomes important so that the sliding of the compacted microparticles isnot hindered and the crevices between vertices of the stellated patterndo not trap microparticles.

At 130, a third pipette tip 133 delivers a wash solution 131 to theprocess chamber 101 containing compacted microparticles. The washsolution may comprise, e.g., a buffer such as Tris-EDTA with asurfactant such as 1% Triton X 100, and having an overall pH 8.0.Typically, the volume of wash buffer is 100 microliters or less, wherethe sample is 2 ml or less in volume. The wash solution is used to washoff any non-DNA and non-RNA molecules, such as inhibitors, that may havebecome bound to the microparticles. The wash solution is chosen topreferentially wash off non-RNA and non-DNA molecules while leaving inplace those RNA and/or DNA molecules bound to the microparticles.Pipette tip 133 may be a different tip from either or both of pipettetips 103 and 123, or may be one of those tips being re-used.

In order to release the RNA and, separately, the DNA from the particles,the wash solution 131 is replaced with an alkaline (pH ˜9.0) releasesolution, e.g., a buffer solution having a pH different from that of thewash solution. This can be done by pipetting out as much of the washsolution as possible, for example, having a residual volume <5microliters, and then dispensing release buffer with a new pipette tip.In case the same tip is used, the liquid should be completely drainedoff so as not to dilute the release solution. For example, at 140, arelease solution 141 is delivered to process chamber 101 so that the RNAbound to the micro-particles can be liberated from thosemicro-particles. In general, the PAMAM(Generation 0) on the particles(as further described herein) most efficiently releases RNA when the pHis about 9. Consequently, RNA can be released from the particles intothe surrounding liquid. In some instances, heat may be applied to theprocess tube, such as to heat the solution to 85° C., to facilitaterelease of the RNA. Generally, the liquid is heated to a temperatureinsufficient to boil liquid in the presence of the particles. In someembodiments, the temperature is 100° C. or less (e.g., less than 100°C., about 97° C. or less). In some embodiments, the temperature is about65° C. or more (e.g., about 75° C. or more, about 80° C. or more, about90° C. or more). In some embodiments, the temperature is maintained forabout 1 minute or more (e.g., about 2 minutes or more, about 5 minutesor more, about 10 minutes or more). In some embodiments, the temperatureis maintained for about 30 minutes (e.g., about 15 minutes or less,about 10 minutes or less, about 5 minutes or less). In some embodiments,the process tube is heated to between about 65 and 90° C. (e.g., toabout 70° C.) for between about 1 and 7 minutes (e.g., for about 2minutes). In other embodiments, the heating is to 85° C. for 3 minutes.In still other embodiments, the heating is to 65° C. for 6 minutes. Ingeneral, a longer heating time is required for a lower temperature.Alternatively, or in combination, particles with retained RNA are heatedto release the RNA without assistance of a release solution. When heatalone is used to release the RNA, the release solution may be identicalwith the wash solution.

Typically, the RNA from a 2 ml sample, and according to the descriptionof the lysis, binding, and washing described elsewhere therein, isreleased into about 20 microliters or less (e.g., about 10 microlitersor less, about 5 microliters or less, or about 2.5 microliters or less)of liquid.

While releasing the RNA has been described as including heating, the RNAmay be released without heating. For example, in some embodiments, therelease solution has an ionic strength, pH, surfactant concentration,composition, or combination thereof that releases the RNA from theretention member without requiring heat.

It is to be noted that excessive shearing, such as is caused by rapidmovements of the liquid during suck-and-dispense mixing operationsduring wash and release (typically during DNA release) in the samplepreparation process may release PAMAM(Generation 0) from the surface ofthe particles, which itself causes downstream inhibition of PCR. Themixing steps should be limited to less than 10 suck-and-dispenseoperations, where the amount moved back and forth ranges from 1-20microliters moved in the pipette, performed over 1-10 seconds persuck-and-dispense operations.

At 150 the microparticles, now having essentially no RNA bound thereto,can be compacted or concentrated in a similar manner to that describedfor 120, but in this case to facilitate removal of the release solutioncontaining the RNA dissolved therein. For example, magnetic beads can becollected together on the interior of the process chamber wall bybringing magnet 121 into close proximity to the outside of the processchamber. In FIG. 1, magnet 121 is used to compact the microparticles atboth stages 120 and 150, though it would be understood that a differentmagnet could be used in both instances.

In instances where a sample contains both RNA and DNA, and it is desiredto determine both a particular RNA and a particular DNA, the proceduresat 140 and 150, as described herein, may be repeated, using a secondrelease solution that is designed to release DNA. As described furtherin U.S. patent application Ser. No. 12/172,208, filed on even dateherewith, and entitled “POLYNUCLEOTIDE CAPTURE MATERIALS, AND METHODS OFUSING SAME”, a solution designed to release DNA typically has a pH ofabout 12 or greater. Such a procedure relies on the fact that RNA andDNA have different pKa's and therefore will elute from the surface of aparticle to which they are non-covalently bound, at different pH's fromone another. Similar considerations, such as release conditions(temperature, reagent concentrations, etc.) apply to release of DNA asto RNA.

It is to be noted that, thus far, all of the processing steps have takenplace in a single tube. This is advantageous for a number of reasons:first, that unnecessary liquid transfer steps will necessarily lead tosome loss of target material. Additional liquid transfer steps will alsoadd to the overall time of the protocol. It should be noted thatperforming all the liquid processing in a single tube is not an easytask primarily because of the residual volumes left between successiveliquid transfers. It becomes even more difficult when the final elutionvolume is very low, such as less than 30 microliters, or less than 20microliters or less than 10 microliters, or less than 5 microliters.Nevertheless, with the protocols described herein, very good yields maybe obtained.

The RNA, and/or subsequently the DNA, liberated from the microparticlescan each be drawn up into a fourth pipette tip 153 in solution in therelease solution. Pipette tip 153 need not be different from all ofpipette tips 103, 123, and 133 and may therefore represent a re-use ofone of those tips. Although it is desirable to use magnetic beads,non-magnetic beads may also be used herein, and separated by, e.g.,centrifugation, rather than by use of a magnet.

In certain embodiments, the ratio of the volume of original sampleintroduced into the processing tube to the volume of liquid into whichthe RNA or DNA is released is at least about 10 (e.g., at least about50, at least about 100, at least about 250, at least about 500, at leastabout 1,000). In some embodiments, RNA or DNA from a sample having avolume of about 2 ml can be retained within the processing tube, andreleased, after binding and washing, into about 4 microliters or less(e.g., about 3 microliters or less, about 2 microliters or less, about 1microliter or less) of liquid.

In some embodiments, the sample has a volume larger than theconcentrated volume of the binding particles having the RNA or DNA boundthereto by a factor of at least about 10.

In other embodiments, the sample has a volume of 100 μl-1 ml, and thecompacted particles occupy an effective volume of less than 2microliters.

The liquid into which the RNA or DNA is released typically includes atleast about 50% (e.g., at least about 75%, at least about 85%, at leastabout 90%, or at least about 95%) of the RNA or DNA respectively presentin the sample 109. Thus, for example, ˜8-10 μg DNA can be liberated from1 ml of overnight culture, and 2-4 μg DNA can be extracted from onebuccal swab. The concentration of RNA or DNA present in the releaseliquid may be higher than the respective concentration in the originalsample because the volume of release liquid is typically less than thevolume of the original liquid sample. For example, the concentration ofDNA in the release liquid may be at least about 10 times greater (e.g.,at least about 25 times greater, at least about 100 times greater) thanthe concentration of DNA in the sample 109. The concentration ofinhibitors present in the liquid into which the RNA or DNA is releasedis generally less than the concentration of inhibitors in the originalfluidic sample by an amount sufficient to increase the amplificationefficiency for the RNA or DNA over that which could be obtained from anunpurified sample.

In general, although the processes and materials described herein arecapable of performing well—usually with only routine adaptation—over awide range of sample sizes, and reagent volumes, for most practicalapplications (considering the size of most biological samples subject todiagnostic analysis), the volume of compacted particles having RNAand/or DNA bound thereto that results (prior to release) is in the range2-3 μl, and is independent of the sample volume, up to about 2 ml ofsample. Typically the quantity of microparticles required is determinedby the quantity of RNA and/or DNA in the sample. It is found that, giventhe efficiency of binding to the particles, 0.5 mg of particles issufficient for most manual applications, and most involving automatedpipetting, regardless of sample size. Thus, for example, for sampleshaving volumes from 0.5 microliters to 3 milliliters, the volume of thecompacted particles is 2-3 μl. For example, for Chlamydia, the samplesize is typically 1 ml, and 0.5 mg of particles is sufficient. For otherapplications, DNA from a 2 ml sample can also be extracted with 0.5 mgparticles, or in some instances 1 mg beads can be used. For smallersamples, such as having a volume of 5 μl, it is still typical to useonly 0.5 mg particles.

In order to agitate the solution at various stages during the manualprocess, the solution may be pipetted up and down a number of times,such as 10 times, 15 times, or 20 times. Such a procedure is acceptableduring the release step as well as the wash steps. Vortexing also worksfor these steps. However, for the automated process, cannot tolerate anymix steps, the number of mixing operations is kept at a minimum as thiswas possibly causing some PAMAM(0) to come off and inhibit downstreamPCR.

The process described herein represents an extremely effective clean-upof a sample in preparation for PCR and provides the capability to detectas few as 25 copies of RNA or DNA from 1 milliliter of clinical sample.The RNA or DNA is present in a high level of concentration because theelution volume can be as low as 3 microliters. There is also a lowresidual sample liquid and/or wash volume in the concentratedmicrospheres, thereby minimizing dilution by sample or wash buffer, aswell as minimizing inhibition from residual sample.

The time interval between introducing the polynucleotide containingsample to processing tube 101, and releasing the RNA or DNA into therelease liquid is usually between 10 and 30 minutes, and is typicallyabout 15-20 minutes, or may be 15 minutes or less (e.g., about 10minutes or less, about 5 minutes or less). These times include the lysistime (which doubles up as a sample-binding time), and are extremelyfast. To release both RNA and DNA, separately, from a single sample, itis only necessary to add an additional release procedure, as in 140 inFIG. 1.

Optionally, at 160 in FIG. 1, the released RNA or DNA in solution may beneutralized by contacting it with a neutralization solution 165 (e.g.,an equal volume of 25-50 mM Tris-HCl buffer pH 8.0). For example, theRNA or DNA in solution in pipette tip 153 may be released into a secondprocess chamber, or vessel, 161 such as a standard laboratory PCR tube,in which the neutralization solution is present. The PCR tube may beremoved and introduced into a PCR machine for further analysis.Typically, the solutions for extracting RNA are close enough toneutrality that a separate neutralization step is not required.

The RNA or DNA in solution in vessel 161 is in a state that it can beamplified, such as by PCR, and detected. Furthermore, the foregoingprocess steps are extremely reliable and robust, and enable quantitativeassays of the extracted RNA or DNA over 7 log dilutions (10-10⁷ copiesof target RNA or DNA/ml of sample).

The process of FIG. 1 has demonstrated effectiveness in manual as wellas automated formats.

The process shown in FIG. 1 may be carried out in conjunction with areagent holder, in which the process chamber may be situated, and inwhich are found appropriate quantities of microparticles, lysissolution, wash solution, release solution, and neutralization solution,each of which is accessible to one or more pipette tips and for use asshown in FIG. 1. An exemplary reagent holder is described in U.S.provisional patent application Ser. No. 60/959,437, filed Jul. 13, 2007,and incorporated by reference herein.

Where a magnet is shown in FIG. 1 for use in compacting magneticmicroparticles, a magnetic separator, as described in U.S. provisionalpatent application Ser. No. 60/959,437, filed Jul. 13, 2007, andincorporated by reference herein, may be used.

Where it is shown in FIG. 1 that heat may be applied to process chamber101, a heater assembly, as described in U.S. provisional patentapplication Ser. No. 60/959,437, filed Jul. 13, 2007, and incorporatedby reference herein, may be used.

The process shown in FIG. 1 is optimally used to prepare highly pure andconcentrated RNA or DNA for use in low-volume (e.g. 4 μl) PCR reactions,such as may be carried out in a microfluidic cartridge, for example amicrofluidic cartridge described in U.S. provisional patent applicationSer. No. 60/959,437, filed Jul. 13, 2007, and incorporated herein byreference.

FIG. 2 shows, schematically, a sample preparation process at themolecular level. At 210, a typical magnetic particle 201, having adiameter of 1 μm, is shown. Attached to the surface of particle 201 aremolecules 205 having a binding affinity for polynucleotides in solutionsurrounding the particle. Attachment of molecules 205 is usually viacovalent bonds. Such molecules are further described herein and in someembodiments are molecules of PAMAM(Generation 0). From 210 to 220, themagnetic particle is incubated in a solution containing RNA and/or DNA,at a pH of 4-8, lower than the pKa of molecules 205. At 220, particle201 is shown having polynucleotide (i.e., DNA and/or RNA) molecules 211attached to the affinity molecules 205. Also shown are various othernon-specifically bound substrates 213, denoted by small ovals,cigar-shapes, and curved lines.

Moving from 220 to 230 in FIG. 2, the particle 201, to which is boundboth RNA and/or DNA molecules 211, and non-specifically bound molecules213, is washed to remove the non-specifically bound substrates, leavinga particle coated in affinity molecules 205 and RNA and/or DNA molecules211 bound thereto. From 230 to 240, the RNA and/or DNA molecules 211 arereleased from the surface of the particle by increasing the pH of thesolution surrounding the particle to a pH of 9 (RNA) and, subsequently apH of 12-13 (to release DNA). The released RNA and/or DNA molecules canbe separately collected in a PCR-ready format.

While samples and various solutions have been described herein as havingmicroliter scale volumes, other volumes can be used. For example,processing tubes with surfaces (e.g., particles) configured topreferentially retain RNA and/or DNA as opposed to inhibitors may havelarge volumes (e.g., many tens of microliters or more, at least about 1milliliter or more). In some embodiments, the processing tube has abench-top scale, and other solutions are correspondingly scaled up.

Polynucleotide Capture Material

Suitable polynucleotide affinity molecules are those that offer a veryhigh density of positively ionizable charges at a low pH, and enablestrong attraction and binding of polynucleotides, including RNA and DNAfrom a clinical lysate, within a few minutes.

A typical embodiment of the materials herein uses: Polyamidoamine(PAMAM) Generation 0, available from the Sigma-Aldrich Chemical Company(“Sigma-Aldrich”), product number 412368. This material, referred tohereinafter as “PAMAM(Generation 0)” or “PAMAM(0)” of “PAMAM(G0)”, is adendrimer whose molecules have the following structure.

The core of the molecule is an ethylene diamine substituted twice onboth nitrogen atoms by an acetyl group. Each acetyl group has itselfreacted with ethylene diamine monomers to yield amino-substituted amidegroups.

The form of PAMAM(0) suitable for use herein is not limited to thatproduct available from Sigma-Aldrich, however. PAMAM(0), beingdendrimeric in nature, admits of a wide range of forms, controlled atleast in part by the extent of dendrimerization permitted during itssynthesis. Thus, many variants of PAMAM(0), having variously, differentnumbers of substituting units, are suitable for use herein. In general,there is a range of sizes of dendrimer molecule (or PAMAM(0) derivative)that is suitable for polynucleotide capture: smaller sizes don't captureenough RNA or DNA, whereas larger sizes retain the RNA or DNA toostrongly, and do not permit easy release. Additionally, differentmonomers from ethylene diamine may be used to make a variant of PAMAMsuitable for use herein. Such monomers may include, without limitation,1,2-propylene diamine, 1,3-propylene diamine, 1,2-butylene diamine,1,3-butylene diamine, and 1,4-butylenediamine.

Molecules of PAMAM suitable for use herein may also be characterized bymolecular weight. In particular, PAMAM(0) has a molecular weight of 516;other suitable PAMAM molecules have weights in the range 500-600 Da.

PAMAM(0) can itself function as an inhibitor of enzymatic processes suchas DNA and RNA amplification, and therefore it is important that it beused in a manner in which it does not reside in solution together withthe released RNA and/or DNA. Aspects of this are further described inthe Examples, hereinbelow.

Support Materials

During use, PAMAM(0) is typically immobilized on, such as bound to thesurface of, a solid support such as carboxylated beads, or magnetic ornon-magnetic beads. In many embodiments, such a solid support comprisesmicroparticles, such as beads, and microspheres. These terms,microparticles, beads, and microspheres may be used interchangeablyherein. The particles are typically formed of a material to which thePAMAM(0) can be easily associated. Exemplary materials from which suchparticles can be formed include polymeric materials that can be modifiedto attach a ligand. Typically, such a solid support itself may bederivatized to yield surface functional groups that react easily withPAMAM(0) molecules to create a chemical bond between the surface and thePAMAM(0). A frequently-employed—and desirable—surface functional groupis the carboxylic acid (COOH) group. Exemplary polymeric materials thatprovide, or can be modified to provide, carboxylic groups and/or aminogroups available to attach PAMAM(0) include, for example, polystyrene,latex polymers (e.g., polycarboxylate coated latex), polyacrylamide,polyethylene oxide, and derivatives thereof. Polymeric materials thatcan used to form suitable particles are described in U.S. Pat. No.6,235,313 to Mathiowitz et al., which patent is incorporated herein byreference. Other materials include glass, silica, agarose, andamino-propyl-tri-ethoxy-silane (APES) modified materials.

During the process of reaction of a PAMAM(0) molecule with acarboxylated particle, such as a magnetic particle, one of the aminegroups out of the total possible amine groups on a PAMAM(0) molecule,such as 6 possible groups in the aforementioned product from SigmaAldrich, is consumed to react with the COOH group of the surface of theparticle to form a carbodiimide bond. (See, e.g., U.S. application Ser.No. 11/281,247, page 40). The remainder of the total number aminegroups, such as 5 groups in the aforementioned product from SigmaAldrich, are available for protonation.

In some embodiments, a synthetic protocol comprises: washing a quantityof microspheres with carbonate and MES buffer; preparing sulfo-NHS andEDAC; incubating the microspheres with sulfo-NHS and EDAC for 30minutes; washing the microspheres with MES and borate buffer; contactingthe microspheres with PAMAM(0) for 8-10 hours; and rinsing unboundPAMAM(0) from the microspheres. An example of synthetic protocols formaking PAMAM(0)-bound microparticles, is given in the Examples,hereinbelow.

There are a variety of sources of bead or particle that can be used tobind PAMAM(0), and used in the processes described herein, for example:Seradyn Magnetic carboxyl modified magnetic beads (Part #3008050250,Seradyn), Polysciences BioMag carboxyl beads, Dynal polymer encapsulatedmagnetic beads with a carboxyl coating, and Polybead carboxylatemodified microspheres available from Polyscience, catalog no. 09850.

The high density of the PAMAM(0) molecules on bead surfaces permits evena small quantity of beads (0.5 mg) to be used for clinical samples aslarge as a milliliter, and permits binding of even low levels of targetRNA or DNA (<100 copies) in a background of billions of copies of otherpolynucleotides.

In some embodiments, at least some (e.g., all) of the particles aremagnetic. In alternative embodiments, few (e.g., none) of the particlesare magnetic. Magnetic particles are advantageous because centrifugationis generally not required to separate them from a solution in which theyare suspended.

Particles typically have an average diameter of about 20 microns or less(e.g., about 15 microns or less, about 10 microns or less). In someembodiments, particles have an average diameter of at least about 4microns (e.g., at least about 6 microns, at least about 8 microns).Magnetic particles, as used herein, typically have an average diameterof between about 0.5 microns and about 3 microns. Non-magneticparticles, as used herein, typically have an average diameter of betweenabout 0.5 microns and about 10 microns.

The particle density is typically at least about 10⁷ particles permilliliter (e.g., about 10⁸ or about 10⁹ particles per milliliter). Forexample, a processing region, such as present in a microfluidic deviceconfigured for used in sample preparation, with a total volume of about1 microliter, may include about 10³ beads.

In some embodiments, at least some (e.g., all) the particles are solid.In some embodiments, at least some (e.g., all) the particles are porous(e.g., the particles may have channels extending at least partiallywithin them).

The microparticles described herein are not only suitable for use inprocess tubes that are handled by manual pipetting operations, but theycan be used in a microfluidic devices, such as in sample concentrator,thereby enabling even sub-microliter elution volumes to be processed, asapplicable.

The microparticles having PAMAM(0) bound thereto are particularlyeffective at capturing, and releasing RNA, and also DNA. In someembodiments, the ratio by weight of the RNA captured by the bindingparticles, to the binding particles prior to contact with the RNA, is5-20%. In other embodiments, the ratio is 7-12%. In still otherembodiments, the ratio is about 10%, corresponding to, e.g., 100 μg ofRNA for each mg of particles.

The microparticles having PAMAM(0) bound thereto are particularlyeffective at capturing RNA, and/or DNA, consistently over a wide rangeof concentrations, thereby permitting quantitative analysis of the RNAand/or DNA to be carried out. In some embodiments, the binding particlescapture 90% or more of the RNA or DNA liberated from cells into asolution in contact with the binding particles, over a range of 1 to 10⁷copies of target RNA or DNA/milliliter of sample.

In some embodiments, the binding particles release 90% or more of theDNA bound thereto when certain release conditions are deployed.

Sample Preparation Kits

Microparticles, coated with PAMAM(0), can be provided to a user in solidform, such as in lyophilized form, or in solution. It is desirable thatthe reagent, however provided, can be used immediately by a user forwhatever intended purpose, without any preparatory steps. Microparticlesprepared by the methods described herein can be lyophilized by methodsknown in the art, and applicable to microparticles of the sizes andcharacteristics described herein.

In each of the kits described herein, neutralization reagents are notrequired in the event that the kits are only to be used for determiningRNA compounds. Thus neutralization reagents may be provided but areoptional. Neutralization reagents are typically deployed in instanceswhen the kits are used for DNA determination or for determining both RNAand DNA.

Such microparticles can also be provided in kit form, in conjunctionwith other reagents that are used, for example, in sample preparation.One embodiment of a kit comprises a number of, such as 24, sealed tubes,each containing lysis buffer; a tube containing lyophilizedmicroparticles having PAMAM(0) bound thereto; a tube containing liquidwash reagents, sufficient to analyze the number of samples; a tubecontaining liquid neutralization reagents, sufficient to analyze thenumber of samples; and a tube containing liquid release reagents,sufficient to analyze the number of samples, wherein each component ofthe kit is stored in an air-tight container. Other numbers of tubesavailable in kit form include 12, 25, 30, 36, 48, 50, 60, and 100. Stillother numbers are also permissible and consistent with the descriptionherein.

Furthermore, in other embodiments of such a kit, the tube containinglyophilized microparticles can additionally contain particles ofreagents selected from the group consisting of: proteinase-k;proteinase-k and mutanolysin; and proteinase-k, mutanolysin, and aninternal control DNA. The additional enzymes are often used incell-specific lysis applications.

In other embodiments, a kit comprises: a first air-tight pouch enclosinga number of—such as 24—tubes, each tube containing lyophilizedmicroparticles having PAMAM(0) bound thereto; a second air-tight pouchenclosing a number of reagent holders, each holder comprising: a tubecontaining liquid lysis reagents; a tube containing liquid washreagents; a tube containing liquid neutralization reagents; and a tubecontaining liquid release reagents. Other numbers of tubes available inkit form include 12, 25, 30, 36, 48, 50, 60, and 100. Still othernumbers are also permissible and consistent with the description herein.

Furthermore, in other embodiments of such a kit, the tube containinglyophilized microparticles can additionally contain particles ofreagents selected from the group consisting of: proteinase-k;proteinase-k and mutanolysin; and proteinase-k, mutanolysin, and aninternal control DNA. The additional enzymes are often used incell-specific lysis applications.

Conditions of DNA Binding and Elution

One factor to consider when assessing the efficacy of a DNA-capturematerial is the material's pKa. The pKa of an acid, HA, is anequilibrium constant for the equilibrium

HA←→H⁺+A⁻,

given by pKa=−log₁₀ Ka, where Ka=[H⁺][A⁻]/[HA]. It can be shown that,when the pH (=−log₁₀ [H⁺]) of the solution is numerically equal to thepKa of the acid, the acid is 50% dissociated at equilibrium. Therefore,knowing the pKa of a material gives an indication of the pH, below whichit is largely dissociated (in anion form), and above which it is largelyunionized.

The pKa for an amino group is defined for its conjugate base, asfollows: a protonated amine, R—NH₃ ⁺ is in dissociative equilibrium:

R—NH₃ ⁺←→⁺+R—NH₂

and its pKa is given by −log₁₀ Ka, where Ka=[H⁺][R—NH₂]/[R—NH₃ ⁺].

Because a nitrogen atom is trivalent, and due to the conditions ofdendrimerization, each molecule of PAMAM(0) has a mixture of primary,tertiary amine groups. Therefore, PAMAM(0) molecules exhibit multiplepK's over a range of values roughly consonant with the range of pKa'sspanned by primary, and tertiary aliphatic amines, whose pKa's typicallylie in the range 10-11, as evidenced by, for example, Table 12.2 ofOrganic Chemistry, 2^(nd) Ed., Allinger, et al., Eds., Worth Publishers,Inc. (1976). However, according to information provided by themanufacturer of PAMAM(0), Dendritech of Midland, Mich., PAMAM is in factlikely to have pKa's in the range of 5.5 (for the tertiary amines in theinterior of the molecule)-9.5 (for the primary amines on the surface ofthe PAMAM molecules). A journal article that references this data isTomalia, et al., Angew. Chem. Int. Ed. Engl., 29, 138-175 (1990), atpage 163, right-hand column.

PAMAM(0) is effective as a binder for DNA in the processes describedherein at least in part because the amine groups of the PAMAM(0) have apKa of between 5-9. Thus, at low pH it is typically positivelycharged—and may even carry multiple positive charges per moleculearising from protonations of the amine groups at pH's lower than itspKa—and is therefore able to bind strongly to polynucleotides such asDNA and RNA, which typically comprise polyanions (are predominantlynegatively charged) in solution.

During the use of the PAMAM(0) molecule in the processes describedherein, the pH of the binding buffer (typically TRIS) used to lyse cellsat the same time as binding liberated DNA to the particles, isapproximately 7-8. At this pH, all the amines (6 possible groups per PEImolecule, as available from Sigma) remain protonated (positivelycharged) and hence strongly attract negative charged DNA molecules tobind towards the beads.

PAMAM(0) molecules are also advantageous because they are resistant to,e.g., are immune to, degradation by lytic enzymes, protease enzymes(e.g., mixtures of endo- and exo-proteases such as pronase that cleavepeptide bonds), harsh chemicals such as detergents, and heat up to 95°C., and as such are able to bind RNA and DNA during the lysis process aswell. Thus, cell lysis and RNA and/or DNA binding can be combined into asingle (synchronous) step, thereby both saving time and at least oneprocessing step. The strong binding of RNA and/or DNA molecules toPAMAM(0) enables rapid washing of affinity beads coated in PAMAM(0) toremove PCR inhibitors using a wash solution. The release of RNA and/orDNA from the affinity beads is effected by an elevation of temperaturein the presence of a proprietary release reagent. As the quantity ofbeads used is very small (<1 μl), the RNA and/or DNA can be released ina final volume as low as 3 microliters. The released RNA and/or DNA isneutralized to a final volume of 5-50 microliters using a neutralizationreagent and is now ready for downstream PCR.

Typically, the amount of sample introduced is about 500 microliters orless (e.g., about 250 microliters or less, about 100 microliters orless, about 50 microliters or less, about 25 microliters or less, about10 microliters or less). In some embodiments, the amount of sample isabout 2 microliters or less (e.g., about 0.5 microliters or less).

PAMAM(0) gives excellent RNA and DNA recovery, based in part on its highbinding capacity, and its high release efficiency. In general, the ratioof mass of particles to the mass of RNA or DNA retained by the particlesis no more than about 25 or more (e.g., no more than about 20, no morethan about 10). For example, in some embodiments, about 1 gram ofparticles retains about 100 milligrams of RNA or DNA; when used insmaller quantities, similar ratios can be obtained (e.g., a bindingcapacity of 100 μg of RNA or DNA/mg beads).

Other Apparatus for DNA Capture

In other embodiments, the solid support can be configured as a retentionmember (e.g., porous member such as a column, filter, a porous membrane,a microporous filter, or a gel matrix, having multiple openings such aspores and/or channels, through which RNA and/or DNA passes) throughwhich sample material (containing the RNA and/or DNA) must pass. Such aretention member may be formed of multiple surface-modified particlesconstrained into a suitable geometry. In some embodiments, the retentionmember comprises one or more filter membranes available from, forexample, Osmonics, which are formed of polymers that may also besurface-modified and used to retain RNA and/or DNA. In some embodiments,a retention member is configured as a plurality of surfaces (e.g., wallsor baffles) across which a sample passes. The walls or baffles aremodified to retain RNA and/or DNA in preference to, e.g., PCRinhibitors. Such a retention member is typically used when themicroparticles are non-magnetic.

As a sample solution moves through a processing region containing such aretention member (suitably modified to preferentially retain RNA and/orDNA), RNA and/or DNA is retained while the liquid and other solutioncomponents (e.g., inhibitors) are less retained (e.g., not retained) andexit the processing region. Typically, such a retention member retainsat least about 50% of RNA and/or DNA molecules (at least about 75%, atleast about 85%, at least about 90%) of the RNA and/or DNA moleculespresent in the sample that entered the processing region. The processingregion is typically at a temperature of about 50° C. or less (e.g., 30°C. or less) during introduction of the sample. Processing can continueby washing the retention member with a wash solution to separateremaining inhibitors from RNA and/or DNA retained by the retentionmember.

In some embodiments, the sample preparation processes described hereinare performed within a microfluidic device, such as a microfluidiccartridge configured to receive a sample, and to capture RNA and/or DNAmolecules from the sample on a solid support contained within it.Exemplary microfluidic cartridges are described in U.S. PatentApplication Publication No. 2006/0166233, and WO2008/061165, both ofwhich are incorporated herein by reference. Such cartridges may includeone or more actuators configured to move microdroplets of various liquidsolutions within the cartridge, a chamber configured to lyse cells inthe sample, and one or more channels and associated valves configured todirect, disrupt, and divert liquid flow within the cartridge.

While sample preparation has been described as being a sequence ofoperations carried out in a single location, such as in a process tubeor a microfluidic cartridge, other configurations can be used. Forexample, in some embodiments, the retention member carrying apolynucleotide-affinity material can be removed from a region where DNAand/or RNA capture occurs, for subsequent processing elsewhere. Forexample, the retention member may be contacted with a mixture comprisingDNA and/or RNA and inhibitors in one location, and then moved to anotherlocation at which the RNA and/or DNA are removed from the retentionmember.

Other Advantages of the DNA Capture Material Described Herein

The extraction reagents and sample preparation processes describedherein offer superior performances compared to currently availableoff-the-shelf kits for sample preparation. Advantages of the materialsand methods herein include the following.

A streamlined sample preparation procedure having fewer steps (as few assix from raw sample to purified RNA and/or DNA) and utilizing fewercontainers than other procedures.

Extraction control (cellular, plasmid, or naked) DNA can also beincluded along with the affinity beads. An internal control DNA can beincluded with the lysis reagents so that the internal control DNA getsco-purified with the other DNA (such as the target DNA) present in theclinical sample, and gets eluted amongst the final released DNA. Duringamplification of the eluted DNA, the internal control DNA is alsoamplified, and can subsequently be detected using a separate fluorophorefrom the target DNA. This gives an extra confirmation that the sampleprep process worked as required.

The description herein has included a characterization of properties anduse of microparticles coated in PAMAM(Generation 0). It would beunderstood by one of ordinary skill in the art that other affinitymolecules may suitably be used in the processes described herein, asdescribed elsewhere (e.g., U.S. patent application publication2006-0166233, incorporated herein by reference).

EXAMPLES Example 1 Sample Preparation Process

The following six steps can be accomplished in as little as 20 minutesfor a single sample, to 30 minutes for a batch of 12 samples, using areagent kit as further described herein. The steps are also easilyautomated as in a system described in U.S. provisional patentapplication Ser. No. 60/959,437, filed Jul. 13, 2007, incorporatedherein by reference. The steps are also shown, schematically, in FIG. 1,and described elsewhere herein.

One exemplary process is as follows.

1. Mix ˜500 μl of the clinical sample with 500 μl of lysis buffer andmagnetic affinity beads, surface-bound with PAMAM(0). Kits for detectingviruses such as EV13 include some lytic enzymes as well dissolved in thelysis buffer.

2. Incubate the mixture of sample, lysis buffer, and beads at atemperature between room temperature and 60° C. for 5-10 minutes, to lysthe cells and bind the RNA and/or DNA to the affinity beads.

3. Separate the magnetic beads and remove as much of the supernatantsolution as possible.

4. Wash the beads with a wash reagent.

5. Release the RNA and/or DNA from the beads by heating the beads for 3minutes at 85° C. in the presence of as little as 3 microliters ofrelease solution.

6. Remove the released RNA and/or DNA and neutralize the solution with aneutralization reagent, such as a Tris buffer, to create PCR-ready RNAand/or DNA.

Another exemplary process is as follows.

-   -   Sample: Mix 500 μl Plasma w 500 μl Prep Buffer, or Dip Swab in 1        mL RNA Prep Buffer.    -   The mixture may optionally be pre-filtered.    -   Incubate @ 60° C. for 10 min.; clean up with proteolytic enzymes        if necessary (swab only) and effect RNA and/or DNA capture by        PAMAM(G0) coated affinity beads (magnetic) as further described        herein.    -   Optionally, in a case where RNA is desired to be determined,        apply DNAse treatment (such as with 7U DNase @ 37 deg C. for 10        min.) to the mixture.    -   Wash RNA bound beads with 100 μl Wash Reagent (2 x) as further        described herein.    -   Release RNA from beads with heat (85° C.; 3 min.) in the        presence of Release Reagent, as further described herein,        thereby liberating RT-PCR ready RNA.

Example 2 Application to a Wide Variety of Matrices

The procedures described herein work for a variety of sample matrices,including both clinical and non-clinical samples, as shown by thefollowing non-exhaustive list:

Nasal swab

CSF

Nasal swab in M4

Nasal swab in UTM

Plasma

Example 3 Representative Results

FIG. 3 shows the use of RNA extraction reagent, PAMAM(0), and a processas further described herein, to isolate and purify Enterovirus 13 (EV13)RNA from Nasal Swabs, using a lysis buffer as described elsewhereherein. The graph shows PCR curves for various samples spiked with eachof 2×10⁴, 2000, 200, 50, and 20 copies/1 mL of RNA Prep buffer. The RNAwas released into 10 μl, but only 2 μl of released RNA was used forRT-PCR, which was performed using a Qiagen RT-PCR kit. PCR curves risefrom the axis in order of decreasing concentration, from left to right.

FIG. 4 shows the use of RNA extraction reagent, PAMAM(0), and a processas further described herein, to isolate and purify Enterovirus 13 (EV13)RNA from Nasal Swabs collected in M4 media. The graph shows PCR curvesfor various samples spiked with each of 1000, 100, and 50, copies/1 mLof sample. PCR curves rise from the axis in order of decreasingconcentration, from left to right.

FIG. 5 shows a comparison of RNA extraction using PAMAM(0) beads betweenbuffer samples and plasma. 500 copies of EV13 RNA per 1 mL were used inboth buffer and plasma samples, according to a process as furtherdescribed herein.

FIG. 6 shows extraction of DNA, using PAMAM(0) beads. 2.5 pg DNA spikedinto an M4 buffer, or a lysis buffer as described herein, were extractedusing a process for extracting RNA from an M4 collection buffer asfurther described herein.

FIG. 7 shows PCR curves for RNA extraction from a 500 μl plasma samplecontaining 200 copies EV13 RNA, using 7U DNAse treatment.

FIG. 8 shows illustrates the sensitivity of the process. Processanalysis reveals an LoD of 50 copies/200 μl CSF.

Example 4 Exemplary Protocol for the Extraction of RNA from M4, Dry Swabin 1×TCEP Buffer, THB Samples

Sample Preparation pre-Processing (only swab samples require filtration)Step Action 1 Pipette 500 μL of specimen into a tube (1.7 ml DOT snapcap tube) containing 500 μL of TCEP buffer 2 Pipette up and down 2x, andthen pipette entire amount into 3 ml syringe 3 Insert plunger intosyringe, and filter contents into a clean tube (1.7 ml DOT snap captube), applying pressure until all liquid is expelled, and foam startsto come out of filter (avoid getting foam into sample).

RNA extraction and PCR prep Step Action 1 Pipette sample (500 μL ofspecimen, plus 500 μL TCEP buffer) into a 1.7 ml DOT snap cap tubecontaining 30 μL of RNA magnetic beads. Cap and invert Reaction Tube 5times, or until beads are dispersed (or dissolved, in the case oflyophilized beads). 2 Immediately place samples in a 60° C. water bath,and incubate for 10 min (10.5 min maximum). 3 Remove samples from waterbath, and dry outsides of tubes with a Absorbent wipe. 4 Place tubes onmagnetic rack, and allow separation to proceed for 1 minute (1.5 minmax). 5 Using a fresh pipette for each sample, carefully aspirate 1 mlof supernatant from each sample (without disturbing beads), using a 1 mlpipettor. Discard supernatant. Be sure to remove any liquid that remainsin the tube cap. 6 After initial removal of supernatants from allsamples, remove any remaining liquid using a fresh 1 ml pipette tip foreach sample. 7 Place tubes in a non-magnetic tube rack, and add 100 μLof Wash Buffer (0.1 mM Tris, pH 7.5) to each tube using a 200 ul pipettetip. Pipette up and down 10 times, or until all magnetic beads areresuspended, and no beads remain stuck to pipette tip. 8 Place tubes onmagnetic rack for 30 seconds, allowing beads to separate. 9 Carefullyaspirate supernatant from all samples using a 200 ul pipette tip.Discard Supernatant. Using a fresh 20 μl tip for each sample, aspirateany remaining liquid left in the sample (ie-liquid that has “settled”following the first aspiration step), and discard the liquid. 10 Placetubes in a non-magnetic tube rack, and add 10 μL of Release Buffer (20mM Bis- Tris Propane or 20 mM Tris pH 9). Vortex for 10 seconds, oruntil beads are resuspended. 11 Place samples in a heat block at 85° C.for 3 minutes (3.5 min max). Remove samples from heat block, and placeon magnetic rack for 30 seconds (1 min max). 12 Remove samples from heatblock, and place on magnetic rack for 30 seconds (1 min max). 13 Keepingtubes on the magnetic rack, remove all liquid, carefully avoidingmagnetic beads on side of tube, and place in 0.65 ml DOT tube. 14 Mixsample by pipetting up and down once. Sample is now ready for PCR. 15Make PCR mix using Quantitect kit spiked with 0.6 uM primers and extraplatinum taq. 16 Add 8 uL of mix into Rotorgene or LC capillaries, add 2μL of RNA. 17 Run RT PCR program as follows: 50 degrees for 20 min (RTstep), 95 degrees for 5 min (denaturation), cycling at 95-2 sec, 58-50sec (50 cycles).

Example 5 Exemplary Protocol for the Extraction of RNA from PlasmaSamples

RNA extraction and PCR prep Step Action 1 Pipette sample (500 ul ofspecimen, plus 500 ul TCEP buffer + 70 uL 10% SDS) into a 1.7 ml DOTsnap cap tube containing 30 ul of RNA magnetic beads. Cap and invertReaction Tube 5 times, or until beads are dispersed (or dissolved, inthe case of lyophilized beads). 2 Immediately place samples in a 60° C.water bath, and incubate for 10 min (10.5 min maximum). 3 Remove samplesfrom water bath, and dry outsides of tubes with a Absorbent wipe. 4Place tubes on magnetic rack, and allow separation to proceed for 1minute (1.5 min max). 5 Using a fresh pipette for each sample, carefullyaspirate 1 ml of supernatant from each sample (without disturbingbeads), using a 1 ml pipettor. Discard supernatant. Be sure to removeany liquid that remains in the tube cap. 6 After initial removal ofsupernatants from all samples, remove any remaining liquid using a fresh1 ml pipette tip for each sample. 7 Add 250 uL DNAse buffer with 1 Dnasepellet or 5 units of liquid DNAse. Resuspend beads by vortexing orpipetting. 8 Incubate at 37 for 10 min. 9 Place tubes on magnetic rack,and allow separation to proceed for 1 minute (1.5 min max). 10 Using afresh pipette for each sample, carefully aspirate 1 ml of supernatantfrom each sample (without disturbing beads), using a 1 ml pipettor.Discard supernatant. Be sure to remove any liquid that remains in thetube cap. 11 Place tubes in a non-magnetic tube rack, and add 100 μl ofWash Buffer (0.1 mM Tris, pH 7.5) to each tube using a 200 ul pipettetip. Pipette up and down 10 times, or until all magnetic beads areresuspended, and no beads remain stuck to pipette tip. 12 Place tubes onmagnetic rack for 30 seconds, allowing beads to separate. 13 Carefullyaspirate supernatant from all samples using a 200 ul pipette tip.Discard Supernatant. Using a fresh 20 ul tip for each sample, aspirateany remaining liquid left in the sample (ie-liquid that has “settled”following the first aspiration step), and discard the liquid. 14 Placetubes in a non-magnetic tube rack, and add 10 μL of Release Buffer (20mM Bis- Tris Propane or 20 mM Tris pH 9). Vortex for 10 seconds, oruntil beads are resuspended. 15 Place samples in a heat block at 85° C.for 3 minutes (3.5 min max). 16 Remove samples from heat block, andplace on magnetic rack for 30 seconds (1 min max). 17 Keeping tubes onthe magnetic rack, remove all liquid, carefully avoiding magnetic beadson side of tube, and place in 0.65 ml DOT tube. 18 Mix sample bypipetting up and down once. Sample is now ready for PCR. 19 Make PCR mixusing Quantitect kit spiked with 0.6 uM primers and extra platinum taq.20 Add 8 uL of mix into Rotorgene or LC capillaries, add 2 uL of RNA. 21Run RT PCR program as follows: 50 degrees for 20 min (RT step), 95degrees for 5 min (denaturation), cycling at 95-2 sec, 58-50 sec (50cycles).

Example 6 Assembly Process for TCEP Buffer for RNA Extractions

The procedure in this example provides a method appropriate forpreparing up to 50 mL of a TCEP Buffer (20 mM Tris HCl pH 7.0, 2%Tx-100, 10 mM TCEP) used in RNA extractions, as further describedherein. The following is a list of reagents utilized in the process.

1 M Tris-HCl pH 7.0

100% Triton X-100 (‘Tx-100’)

TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)

Ultrapure Water

The following is a list of equipment utilized in the process.

Laminar flow hood

Serological pipette filler

Serological pipette

Vortexer

Appropriate size container

New, sterile graduated cylinder

Appropriate Personal Protective Equipment (PPE)

Product Label

An operator performing this procedure must know how to prepare buffers,and possess an excellent pipetting technique, and should exercisegeneral lab sterile techniques, prepare the solution in a laminar flowhood for sterility, and be cautious to not contaminate stock reagents.Gloves and lab coat should be worn by operator at all times.

Preparation of 50 mL of the 2x TCEP Buffer (20 mM Tris HCl pH 7.0, 2%Tx-100, 10 nM TCEP) Step Action For 50 mls 1 Verify reagent availabilityand expiration. Also perform a visual inspection of stock reagents. 2Affix the product label to the appropriate container. 3 Using aserological pipette or a new graduated cylinder dispense 45 mL H20ultrapure water into the container. 4 Using a serological pipette or anew graduated cylinder, 1 mL Tris-HCl 1M Tris-HCl dispense 1M Tris-HClpH 7.0 into container. 5 Weigh out appropriate amount of TCEP and add tocontainer. 143 mg 6 Vortex/shake well to mix. Do not add Triton untilthe TCEP has completely dissolved. 7 Using a serological pipette, addTriton X-100 to container being careful 4 mL to get all of solution outof pipette. 8 Vortex/shake well to mix. 9 Store at room temperature.

Example 7 Exemplary Process for the Preparation of Magnetic RNA AffinityMicrospheres

This procedure provides an appropriate method for one batch of PAMAM(G0)coated magnetic microspheres, commonly referred to as MagneticRNA-Affinity Microspheres. One batch consists of 1-10 ml synthesisresulting in 6 mL of magnetic RNA affinity microspheres. A flow-chart ofthe process is shown in FIG. 9. The following is a list of equipmentutilized in the process.

Vortexer

Microcentrifuge

Magnetic Rack

1.7 mL microcentrifuge tubes

4-oz specimen containers

50 mL conical tubes

15 mL conical tubes

Centrifuge

pH meter

Pipettors

Pipettor tips

Ultrasonic dismembrator

dH20 wash bottle

Task Wipers

Balance

Laboratory marker

Gloves and Labcoat

Orbital shaker

Labeling tape

Pipette filler

Serological pipettes

An operator performing this procedure must be competent with amicrobalance, pipettors, pH meter, ultrasonic dimembrator and amicrocentrifuge, and must know how to prepare buffers and possess anexcellent pipetting technique. Gloves, labcoat, and eye protectionshould be worn by the operator at all times. Ear protection must be wornduring sonication steps. All solutions are prepared in a laminar flowhood.

Procedure - Preparation of Buffers Step Action 1 Verify availability andcheck expiration dates of all solutions and reagents. 2 Visually inspectall stock solutions and reagents for precipitation or color change. Donot use if precipitation occurs or color changes. 3 Equilibrate allaliquoted stock solutions and reagents to RT. Take out EDAC and NHS from−20° C. and equilibrate to RT before use, this should take approximatelyone hour.

Preparation of 70 mL Buffer SN-B (50 mM MES Buffer pH 6.1, 0.15% TritonX-100) Step Action For 70 mls 1 Label a 4-oz or 500 ml container with“Buffer SN-B”, date, and initials. 2 Using a serological pipette BufferSN-C to bottle. 7 mL 3 With a P5000, 10% Triton X-100 to bottle. 420 uL4 Using a graduated cylinder, add ultrapure water to bottle. 62.5 mL 5Mix well by inversion. 6 Check pH of solution, which should be between5.8-6.5. 7 Store at 4° C. during overnight incubation but discard afterlot manufacture is complete.

Preparation of 50 mL Buffer SN-G (50 mM Tris pH 7.5, 0.1% Triton X-100)Step Action For 50 mls 1 Labe a 50 mL conical tube with “Buffer SN-G”,date, and initials 2 Using a serological pipette add 1M Tris pH 7.5 tobottle 2.5 mL 3 With a P5000, 10% Triton X-100 to bottle. 200 uL 4 Usinga graduated cylinder, add ultrapure water to bottle. 47.3 mL 5 Mix wellby inversion. 6 Chek pH of solution, which should be between 7.2-7.7 7Store at RT for the duration of lot manufacture but discard after lotmanufacture is complete.

Preparation of 10 mL Buffer SN-H (50 mM MES pH 6.1, no Tx) Step ActionFor 10 mls 1 Label a 15 mL conical tube with “Buffer SN-H”, date, andinitials. 2 Using a serological pipette add Buffer C to bottle 1 mL 3Using a graduated cylinder, add ultrapure water to bottle. 9 mL 4 Mixwell by inversion. 5 Check pH of solution, which should be between5.8-6.5 6 Store at RT for the duration of lot manufacture but discardafter lot manufacture is complete.

Steps to be performed on Day 1, include the following.

Step Action 1 Calculate required amount of carboxylated microspheres.Divide 10 mL by % solids to calculate amount of microspheres needed perreaction. 5 reactions per set. Multiply this number by 5 to get thetotal amount of microspheres for the full set. 2 Vortex the vialcontaining the microspheres very well (for approx 1 minute). 3 Label1-50 mL conical tubes with Lot number, date, and initials. 4 Pipette theappropriate amount of microspheres into 50 mL conical tube. 5 Placeconical tubes onto magnetic rack and let sit until beads are fullycaptured by magnet. Remove supernatant carefully. MES buffer wash For 30mls 6 Add Buffer SN-B to each tube and vortex to mix 10 mls Placeconical tubes onto magnetic rack and let sit until beads are fullycaptured by magnet. Remove supernatant carefully. Repeat wash 2 moretimes. Prepare sulfo-NHS 7 Weigh out small amount of sulfo-NHS on weighpaper and multiply weight (in mg) by 20 to calculate μL of ultrapurewater to add to make 50 mg/ml solution. Need 1.5 mL for 1 reactions (75mg). Add to 1.7 mL tube and mix well. Weight (mg) × 20 = μL ultrapurewater needed. Add ultrapure water and vortex well to resuspend. NHSsolution should be prepared right before use, discard after 15 min. For30 mls Activation (Prepare EDAC in hood) 8 Add reagents in the followingorder to each conical tube: For 30 mls (i) ddH₂O 5000 μL (ii) BufferSN-C 1000 μL (iii) 50 mg/mL sulfo-NHS 1500 μL Sonicate using ultrasonicdismembrator at a power output of 12 10 seconds for the appropriate timemaking sure that the probe is submerged at all time. Clean probe withdH₂O and wipe with absorbent wipe before and after sonication. 9Immediately prepare 5 mg/ml EDAC in hood. EDAC solution should beprepared right before use, discard after 15 min. Weigh out small amountof EDAC onto weigh paper and multiply weight (in mg) by 200 to calculateμL of ultrapure water needed to make 5 mg/ml solution. Need 2480 μltotal (12.4 mg). Prepare in 50 mL conical tube. Weight (mg) × 200 = (μL)ultrapure water to add. Vortex well after addition of ultrapure H2O. 10Add the following: (i) 10% Triton X-100 10 μL 10 μL (ii) 5 mg/mL EDAC(add EDAC solution carefully; drop by 2480 μL drop 2480 μL whilevortexing the solution at very low speed) (iii) 50 mg/mL sulfo-NHS 1500μL Mix well by vortexing. 11 Record number of times EDAC opened onbottle. Discard after 5 uses. 12 Secure tubes to orbital shaker withlabeling tape and incubate at 30 mins. room temperature at setting 6 (orat setting where microspheres are mixing well). 13 After incubation,centrifuge for 5 min at maximum speed and then 5 mins. place on magnet.Remove supernatant carefully but completely. MES buffer wash For 30 mls14 Add Buffer SN-B to each tube and vortex to mix 10 mls Place conicaltubes onto magnetic rack and let sit until beads are fully captured bymagnet. Remove supernatant carefully. Repeat wash 2 more times. Coupling15 Prepare coupling reaction: For 10 mls (i) Add Buffer H 6 mL Sonicateusing ultrasonic dismembrator at power output 9 for the 10 secappropriate time making sure that the probe is submerged. Clean probewith dH₂O and wipe with absorbent wipe before and after sonication. (ii)Add PAMAM(G0) (add solution carefully, drop by drop 250 μL whilevortexing the solution on low speed). (iii) Mix by vortexing 16 Securetube to orbital shaker with labeling tape. Incubate overnight at asetting of 6 at room temperature (or at setting where microspheres aremixing well). 17 Store buffers SN-B, H and G at 4° C. overnight. ReturnNHS and EDAC stocks to −20° C. Return buffer SN-C to 4° C.

Steps to be performed on Day 2, include the following.

Step Action 18 After overnight incubation, remove buffers SN-B and Gfrom 4° C. and equilibrate to RT (approximately 1 hr). 19 Centrifugetubes for 5 min at maximum speed and place on magnetic rack. Removesupernatant carefully but completely. Tri Washes For 30 mls 20 AddBuffer SN G to each tube and vortex to mix. Clean probe 10 mL with dH20and wipe with absorbent wipe before and after sonication. Place conicaltubes onto magnetic rack and let sit until beads are fully captured bymagnet. Remove supernatant carefully. Repeat wash 2 more times. FinalResuspension 21 Resuspend each reaction in Buffer SN-B by sonicationusing 6 mL ultrasonic dismembrator at power output of 12 for 10 seconds(ensure that probe is submerged). Clean probe with dH2O and wipe withabsorbent wipe before and after sonication. 22 Affix appropriate labelto tube. 23 Discard all buffers. Store Buffer SN-C at 4° C. 25 Store at4° C. Stable for 1 month if stored appropriately.

The foregoing description is intended to illustrate various aspects ofthe instant technology. It is not intended that the examples presentedherein limit the scope of the appended claims. The invention now beingfully described, it will be apparent to one of ordinary skill in the artthat many changes and modifications can be made thereto withoutdeparting from the spirit or scope of the appended claims.

1-12. (canceled)
 13. A method for isolating polynucleotides from acell-containing sample in a process tube, the method comprising: lysingthe cells in the sample by contacting the sample with a lysis solutionhaving a pH between 4 and 8, thereby releasing polynucleotides from thecells; retaining the polynucleotides in the sample as compared to anyamplification inhibitors by contacting the sample with magnetic bindingparticles having PAMAM(Generation 0) molecules covalently bound to thesurface thereof, the polynucleotides becoming reversibly retained by thePAMAM(Generation 0) molecules on the binding particles; compacting thebinding particles retaining the polynucleotides in the process tube bypositioning a magnet in communication with the binding particles;removing the lysis solution from the process tube; washing the bindingparticles with a wash solution having a pH less than 9 while thepolynucleotides are retained on the binding particles; and releasing thepolynucleotides from the binding particles by contacting the bindingparticles with a release solution having a pH between 9 and
 12. 14. Themethod of claim 13, wherein the step of lysing the cells furthercomprises heating the lysis solution in the process tube.
 15. The methodof claim 13, wherein the step of releasing the polynucelotides furthercomprises heating binding particles in the process tube.
 16. The methodof claim 13, wherein the steps of lysing, retaining, compacting,removing, washing, and releasing all take place in a single processtube.
 17. The method of claim 13, wherein the release solution has avolume of less than 3 microliters.
 18. The method of claim 13, whereinthe release solution comprises Tris.
 19. The method of claim 13, whereinthe release solution comprises Bis-Tris Propane.
 20. The method of claim13, wherein the release solution has a pH between 9 and
 10. 21. Themethod of claim 13, wherein the release solution has a pH between 10 and11.
 22. The method of claim 13, wherein the release solution has a pHbetween 11 and
 12. 23. The method of claim 13, wherein the releasesolution has a pH of approximately
 9. 24. The method of claim 13,wherein the release solution has a pH of approximately
 10. 25. Themethod of claim 13, wherein the release solution has a pH ofapproximately
 11. 26. The method of claim 13, wherein the releasesolution has a pH of approximately
 12. 27. A method for isolatingpolynucleotides in a sample, comprising: contacting a solution of thesample with a plurality of binding particles, the binding particleshaving polycationic dendrimer molecules covalently bound to carboxylgroups on the surface of the binding particles, wherein thepolynucleotides become reversibly retained by the dendrimer molecules ascompared to any amplification inhibitors in the solution of the sample,the amplification inhibitors remaining in the solution; removing thesolution containing the amplification inhibitors from the presence ofthe binding particles; washing the binding particles while thepolynucleotides are retained by the binding particles; and releasing thepolynucleotides from the binding particles with a release solutionhaving a pH between 9 and
 12. 28. The method of claim 27, wherein thedendrimer molecules are PAMAM molecules.
 29. The method of claim 28,wherein one amine group on the PAMAM molecules covalently bonds with thecarboxyl groups on the surface of the binding particle.
 30. The methodof claim 28, wherein a plurality of amine groups on the PAMAM moleculescovalently bond with the carboxyl groups on the surface of the bindingparticle.
 31. The method of claim 28, wherein the PAMAM molecules on thebinding particles have a molecular weight in the range of 500-600 Da.32. The method of claim 28, wherein the PAMAM molecules on the bindingparticles comprise PAMAM(Generation 0) molecules.
 33. The method ofclaim 32, wherein the PAMAM molecules on the binding particles have amolecular weight of approximately 516 Da.
 34. The method of claim 32,wherein the PAMAM molecules on the binding particles comprise five aminegroups available for protonation.
 35. The method of claim 32, whereinthe PAMAM molecules on the binding particles comprise less than fiveamine groups available for protonation.